The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.
Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.
Adult neurogenesis is a biological process by which new neurons are born in an adult and integrated into the existing neural networks1. In humans, this process occurs in the dentate gyrus (DG) of the hippocampus, where about 1,400 new cells are born each day2. These cells reside in the inner part of the DG, which harbors a neurogenic niche, termed the subgranular zone (SGZ). Here, hippocampal adult neural stem cells (NSCs) undergo a complex developmental process to become fully functional neurons that contribute to the regulation of specific brain functions, including learning and memory, mood regulation, and stress response3,4,5,6. To influence behaviors, adult NSCs are highly regulated by various external stimuli in an activity dependent manner by responding to an array of local and distal chemical cues. These chemical cues include neurotransmitters and neuromodulators and act in a circuit specific manner from various brain regions. Importantly, circuit wide convergence of these chemical cues on NSCs allows for unique and precise regulation of stem cell activation, differentiation, and fate decisions.
One of the most effective ways to interrogate circuit regulation of adult NSCs in vivo is by pairing immunofluorescence analysis with circuit wide manipulations. Immunofluorescence analysis of adult NSCs is a commonly utilized technique, where antibodies against specific molecular markers are used to indicate the developmental stage of adult NSCs. These markers include: nestin as a radial glia cell and early neural progenitor marker, Tbr2 as an intermediate progenitor marker, and dcx as a neuroblast and immature neuron marker7. Additionally, by administering thymidine analogs such as BrdU, CidU, Idu, and Edu, cell populations undergoing S phase can be individually labeled and visualized8,9,10. By combining these two approaches, a wide range of questions can be investigated ranging from how proliferation is regulated at specific developmental stages, to how various cues affect NSC differentiation and neurogenesis.
Several options exist to effectively manipulate neural circuits including electrical stimulation, optogenetics, and chemogenetics, each with their own advantages and disadvantages. Electrical stimulation involves an extensive surgery where electrodes are implanted to a specific brain region which are later used to transmit electrical signals to modulate a targeted brain region. However, this approach lacks both cellular and circuit specificity. Optogenetics involves the delivery of viral particles that encode a light activated receptor that is stimulated by a laser emitted through an implanted optical fiber, but requires extensive manipulations, large cost, and complex surgeries11. Chemogenetics involves the delivery of viral particles that encode a designer receptor exclusively activated by designer drugs or DREADDs, which are subsequently activated by a specific and biologically inert ligand known as clozapine N-oxide (CNO)12. The advantage of utilizing DREADDs to manipulate local neural circuits that regulate adult NSCs lies in the ease and various routes of CNO administration. This allows for a less time-consuming approach with reduced animal handling, which is easily adaptable for long term studies to modulate neural circuits.
The approach described in this protocol is a comprehensive collection of various protocols required to successfully interrogate circuit regulation of adult hippocampal neurogenesis that combines both immunofluorescence techniques and circuit manipulations using chemogenetics. The method described in the following protocol is appropriate for stimulating or inhibiting one or multiple circuits simultaneously in vivo to determine their regulatory function on adult neurogenesis. This approach is best used if the question does not need a high degree of temporal resolution. Questions requiring precise temporal control of stimulation/inhibition at a certain frequency, can be better addressed using optogenetics13,14. The approach described here is easily adapted for long term studies with minimal animal handling especially where stress is a major concern.
All procedures including animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina Chapel Hill.
1. Stereotaxic Injection of Viral Particles
2. Clozapine N-oxide Administration
3. Thymidine Analog Labeling
4. Tissue Preparation and Processing
Antifreeze Solution | Ethylene-glycol 150 mL + sucrose 150 g + fill to 500 mL 0.1 M PB for 500 mL solution |
Citrate Buffer | 9 mL of citric acid stock + 41 mL of tri-sodium citrate buffer + 450 mL of ddH2O |
Citric acid stock | [0.1 M] Citric Acid 21 g/1 L ddH2O |
Tri-sodium citrate stock | [0.1 M] Tri-sodium Citrate 29.4 g/1 L ddH2O |
Tris Buffered Saline -Triton (TBS -Triton) | 0.05% 100-x Triton in TBS |
Permeabilization Buffer | 0.5% 100-x Triton in TBS |
Blocking Buffer | 0.33 mL Donkey Serum in 10 mL TBS-Triton |
Edu Reaction Solution | Make a CuSO4·5H2O solution by adding 1 mg of CuSO4·5H2O in 4 mL solution of [0.1 M] Tris pH 8.5. Then add 1:40 of a 600 µM Alexa488-azide solution and 10 mg/mL of L-Na+ ascorbate to the CuSO4·5H2O solution before applying to tissue. |
Table 1: Solutions utilized for immunohistochemistry.
5. Immunohistochemistry
6. Image Collection
7. Image Analysis
Following the experimental procedures described above (Figure 1A,B), we were able to determine the effects of stimulating contralateral mossy cell projections on the neurogenic niche within the hippocampus. By utilizing a Cre-dependent Gq-coupled stimulating DREADD virus paired with a mossy cell labeling 5-HT2A Cre-line, we were able to selectively activate excitatory projections from mossy cells onto the contralateral DG and determined that strong mossy cell stimulation promoted stem cell quiescence (Figure 1C). We verified accurate viral delivery before the analysis of tissue (Figure 2A,B). Additionally, we verified activation of mossy cells via c-fos immunohistochemistry experiments (data not shown). In the case of improper viral injection, exclude animal from further analysis. An improper injection is one that fails to target the desired coordinates, has most of the expression outside the desired region, or has little to no viral delivery. For this experiment, mossy cells in the hilus of the DG were the intended target, and if injections were outside of the hilus, they were excluded. By using a thymidine analog, Edu, and antigen retrieval for the nestin staining outlined in sections 5.2 and 5.3, we were able to successfully label proliferating neural stem cells (Figure 3A). Additionally, by omitting the antigen retrieval step, section 5.2, we were able to label Tbr2 positive neural progenitor and neuroblast, and DCX positive neuroblast and immature neurons (Figure 3A). We demonstrate an example of the area quantified and used to calculate density and provide an example of mounted tissue on a slide (Figure 3B and Figure 4A). Moreover, both successful and sub-par experiments are provided as references for experimental approaches (Figure 4B). Lastly, there are several different quantifications that can be obtained from a successful experiment (Figure 5A-D)15. The quantifications include the density of proliferating neural stem cells (Nestin+/Edu+/volume), the percent of proliferating neural stem cells (Nestin+/Edu+/total nestin), total proliferating cells (Edu+/volume) and total stem cell pool (Nestin+/volume). Upon contralateral stimulation of a mossy cells, a decrease in neural stem cell proliferation was observed. Similar quantifications can be obtained for neural progenitor and immature neurons by using the appropriate antibody.
Figure 1: Experimental approach to assay circuit regulation of adult neural stem cells. (A) Schematic representing the different steps outlined in the protocol. (B) Timeline of experimental approach used to stimulate mossy cells in rodents. (C) Injection schematic targeting contralateral mossy cells for stimulation. (D) Schematic of the developmental lineage of adult neural stem cells in the subgranular zone (SGZ), granule cell layer (GCL), and molecular layer (ML) with corresponding antibodies used at different developmental stages. The different developmental stages include either quiescent or activated radial neural stem cells (NSC), neural progenitors (NP), neuroblast (NB), immature granule cells (GC), and mature GC. Please click here to view a larger version of this figure.
Figure 2: Demonstration of effective viral delivery. (A) Immunofluorescence images of accurate viral delivery. Viral particles express an mCherry fluorescent label which target mossy cells in the hilus (white boxed region). DAPI labels cell nuclei. The image side of accurate viral delivery demonstrates clear mossy fiber projections from the contralateral injection side. (B) Immunofluorescence images of off target viral injections. Note that in this case contralateral mossy fibers are absent in the image side. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 3: Analysis of proliferating neural stem cells and progeny. (A) Immunohistochemistry images of thymidine analog Edu colocalizing with specific cell stage markers nestin (neural stem cells and progenitors), Tbr2 (neural stem cells, neural progenitors), and DCX (neuroblast, immature neurons) indicated by white arrowheads. Scale bar = 10 µm. (B) Representative measurement of the area within the dentate gyrus used to calculate density. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 4: Demonstration of immunofluorescence preparation. (A) Schematic demonstrating stereological separation of mounted tissue sections from anterior to posterior axis. Red box denotes the side imaged. (B) Demonstration of successful and sub-par immunofluorescence experiments for neuronal lineage markers. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 5: Contralateral activation of mossy cells decreases neural stem cell proliferation. (A) Immunohistochemistry quantifications of Nestin+/Edu+ cells in the dentate gyrus demonstrate a decrease in proliferation after stimulation of contralateral mossy cells. (B) Decrease in the percent of proliferating neural stem cells in the group with activated contralateral mossy cells. (C) There was no significant change in neural stem cell density in either group. (D) There was no change in overall levels of proliferating cells in the dentate gyrus. Values represent means ± standard error of measurement. p < 0.05 (n = 3 for control, n = 5 for the hM3D group). This figure has been adapted from Yeh et al.15. Please click here to view a larger version of this figure.
The goal of this protocol is to assess how manipulating specific neural circuits regulates adult hippocampal neurogenesis in vivo using a series of immunohistochemistry techniques. Assaying activity dependent regulation of adult neurogenesis mediated by specific neural circuits is a valuable technique with great potential for modifications to study a diverse range of neural circuits. The success of these types of experiments depends on multiple factors including accurate viral delivery, proper viral selection for the desired manipulation, proper delivery of a thymidine analog, animal age, immunostaining efficiency, successful transcardial perfusions, and unbiased quantification of images. For example, inaccurate viral delivery may cause off target effects that result in a phenotype unrelated to the circuit in question. Additionally, low quality immunofluorescence techniques may hide the true number of present cells and therefore produce a phenotype that is not biologically relevant. Another very important factor to control is the age of the mice when performing experiments, considering that adult neurogenesis is age dependent22. Lastly, it is important that each section is unbiasedly scored. To reduce bias, take a methodical approach and ensure that the person scoring is proficient at identifying the stages of adult NSC development using morphological information. Additionally, blind both control and treatment groups and reveal their identities after image quantifications. As an additional measure to reduce bias, two separate individuals can quantify the same data set to validate observed results.
There are several limitations associated with this approach to study circuit activity dependent regulation of adult NSCs and newborn progeny. The first limitation is that this approach does not provide information about the specific cell types within a circuit that mediate the overall effect on NSCs from manipulating the circuit in question. This means that although there might be a phenotypic effect on adult NSCs, the effect may be acting through one or several intermediate cell types. An efficient way to address this concern is to pair these studies with electrophysiology to pin down the intermediaries. An additional limitation of this protocol is the need to have either a specific Cre mouse (5-HTR2A) line or a viral construct (AAV5-camKII-hM3d-mcherry) that can target the desired circuit. If an effective cell specific Cre mouse line is not readily available for a question of interest, the ability to study this circuit becomes increasingly difficult. However, many cell types in the brain have Cre specific mouse lines. A lesser limitation of this protocol is related to CNO as an effective inert ligand. Recently, studies demonstrated that CNO, the inert chemical used to activate DREADDs, metabolizes to clozapine, which may cause behavioral phenotypes23. However, an efficient way to address is to include proper controls in each experiment. An example of proper controls includes both a CNO and DREADD control, where CNO is administered in combination with a control reporter virus (AAV5-DIO-mCherry), and a saline only control where no CNO is administered to a reporter virus group. By including these controls, the effects of only CNO can be isolated. Alternatively, a secondary inert ligand known as C21, has been recently demonstrated to have similar efficacy and potency with no demonstrated behavioral effects24. Lastly, a final limitation of this protocol is controlling the amount of CNO that each animal consumes during the experiment. Different animals drink CNO-containing water at varying degrees and may therefore have a range of effects on adult neurogenesis. In general, a mouse tends to drink about 4 mL of CNO water in a 24 h period. This means that at the concentration of 1 mg/200 mL animals receive a total of 0.02 mg of CNO per day which is comparable to the amount of a single CNO dose injected intraperitoneally. If timely coupled administration of CNO is a concern, switching to intraperitoneal injections may be a better alternative.
The advantage of using this protocol is the degree of specificity achieved when modulating adult neurogenesis. Past neurogenesis studies have utilized systemically administered pharmacological agonist or antagonist to modulate circuit components. These non-specific manipulations may produce phenotypic differences but provide little insight about mechanisms involved in adult neural stem cell regulation. Additionally, this protocol can be easily modified to investigate various circuit wide effects on adult neurogenesis. For example, by switching to an inhibitory DREADD, or by targeting one or multiple brain regions at once, one can ask an array of questions to understand circuit specific regulation of adult neurogenesis. Another advantage of using this protocol over previous approaches is that, the use of a nestin antibody eliminates transgenic animal breeding of fluorescently encoded neural stem cell reporters such as nestin:GFP, increasing efficiency and reducing time per experiment8. Furthermore, this technique limits rodent handling when administering CNO, which reduces rodent stress during experiments. It is important to mitigate stress when studying stress-sensitive processes. Lastly, this approach is easily amendable to include a behavioral assay, for example, if one were interested in asking if the contralateral mossy cell circuit that modulates NSCs also plays a role in spatial learning or stress resilience.
The main technical difficulty when using this approach is accurate viral delivery. Becoming a proficient rodent surgeon takes practice and can take significant troubleshooting. It is therefore advisable to perform a series of pilot experiments to test viral titer, labeling efficiency, and viral spread. We have found that certain serotypes have different spreading patterns and that the AAV2 serotype spreads less than AAV5 or AAV8. Additionally, it is best to have a trusted viral packaging provider for each of these experiments. By performing pilot surgeries, many of these concerns can be addressed and one can save time. It is also recommended that one test different CNO concentrations to stimulate or inhibit the desired circuits. In general, 1 mg/kg will sufficiently activate tested circuits, but certain cell types may require more or less CNO. It is important to note that the dose of CNO administration can differentially affect certain circuits specifically when looking at something such as mossy cells15.
Alternative applications of this protocol include simultaneous behavioral testing, modulation of alternative circuits, and additional analysis of neurogenesis features. To perform behavioral testing, one could follow the protocol described and after administering CNO, perform a specific behavioral task, such as a novel location assay, or a spatial navigation task. The benefit of this approach is that a single experiment would yield both behavioral and circuit specific information that could lead to a circuit specific behavioral phenotype. To modulate alternative circuits, one can use a combination of different Cre lines and viral vectors. For example, if one were interested in understanding how inhibiting dopaminergic neurons from the ventral tegmental area (VTA) or VTA modulates adult neurogenesis, one could use a tyrosine hydroxylase Cre mouse line and inject a Cre dependent hM4D (inhibitory) DREADD virus in to the VTA to determine dopaminergic specific regulation of adult neurogenesis. The possibilities to target alternative brain regions using this approach are vast and can be strategically used to interrogate compelling neural circuits. Lastly, this approach allows one to investigate additional stages of adult neurogenesis. If for example one wanted to understand how stimulating mossy cells affects arborization or dendritic length of immature neurons, one would follow a similar protocol but perform alternative analysis such as sholl analysis.
In summary, this protocol provides a detailed step-by-step process to assay circuit activity dependent regulation of adult NSCs and neurogenesis via DREADD technology. The strength of this protocol lies in its ability to be easily modified to address a vast range of questions regarding circuit specific adult neural stem cell regulation. With the advancement of the clustered regularly interspaced short palindromic repeats (CRISPR) technology, it is now easier to generate cell specific Cre mouse lines to pair with sophisticated viral constructs to address increasingly complex questions expanding the applicability of this protocol.
The authors have nothing to disclose.
L.J.Q. was supported by the National Institute of Mental Health of the National Institutes of Health under Diversity Supplement R01MH111773 as well as a T32 training grant T32NS007431-20. This project was supported from grants awarded to J.S. from NIH (MH111773, AG058160, and NS104530).
24 Well Plate | Thermo Fisher Scientific | 07-200-84 | |
48 Well Plate | Denville Scientific | T1049 | |
5-Ethynyl-2'-deoxyuridine (Edu) | Carbosynth | NE08701 | |
Alcohol 70% Isopropyl | Thermo Fisher Scientific | 64-17-5 | |
Alcohol Prep Pads | Thermo Fisher Scientific | 13-680-63 | |
Alexa-488 Azide | Thermo Fisher Scientific | A10266 | |
Anti-Chicken Nestin | Aves | NES; RRID: AB_2314882 | |
Anti-Goat DCX | Santa Cruz | Cat# SC_8066; RRID: AB_2088494 | |
Anti-Mouse Tbr2 | Thermo Fisher Scientific | 14-4875-82; RRID: AB_11042577 | |
Betadine Solution (povidone-iodine) | Amazon | ||
Citiric Acid Stock [.1M] Citric Acid (21g/L citric acid) | Sigma-Aldrich | 251275 | |
Clozapine N- Oxide | Sigma-Aldrich | C08352-5MG | |
Confocal Software (Zen Black) | Zeiss Microscopy | Zen 2.3 SP1 FP1 (black) | |
Copper (II) Sulfate Pentahydrate | Thermo Fisher Scientific | AC197722500 | |
Cotton Swabs | Amazon | ||
Coverslip | Denville Scientific | M1100-02 | |
Delicate Task Wipe Kimwipes | Kimtech Science | 7557 | |
Drill Bit .5mm | Fine Science Tools | 19007-05 | |
Ethylene Glycol | Thermo Fisher Scientific | E178-1 | |
Hamilton Needle 2 inch | Hmailton Company | 7803-05 | |
Hamilton Syringe 5uL Model 75 RN | Hmailton Company | Ref: 87931 | |
High Speed Drill | Foredom | 1474 | |
Infusion Pump | Harvard Apparatus | 70-4511 | |
Injectable Saline Solution | Mountainside Health Care | NDC 0409-4888-20 | |
Insulin Syringe | BD Ultra-Fine Insulin Syringes | ||
Isoflurane | Henry Schein | 29405 | |
Stereotax For Small Animal | KOPF Instruments | Model 942 | |
Leica M80 | Leica | ||
Leica Microtome | Leica | SM2010 R | |
LSM 780 | Zeiss Microscopy | ||
Nair (Hair Removal Product) | Nair | ||
Paraformaldahyde 4% | Sigma-Aldrich | 158127 | |
Plus Charged Slide | Denville Scientific | M1021 | |
Phosphate Buffered Solution (PBS) | Thermo Fisher Scientific | 10010031 | |
Puralube Vet Ointment | Puralube | ||
Slide Rack 20 slide unit | Electron Microscopy Science | 70312-24 | |
Slide Rack holder | Electron Microscopy Science | 70312-25 | |
Small Animal Heating Pad | K&H | ||
Sucrose | Sigma-Aldrich | S0389 | |
Super PAP Pen 4 mm tip | PolySciences | 24230 | |
Surgical Scalpel | MedPride | 47121 | |
Tris Buffered Solution (TBS) | Sigma-Aldrich | T5912 | |
Tri-sodium citrate Stock [.1M] Tri-sodium Citrate (29.4g/L tri-sodium citrate) | Sigma-Aldrich | C8532 | |
Triton X-100 | Sigma-Aldrich | 93443 | |
Tweezers | Amazon | ||
Vet Bond Tissue Adhesive | 3M | 1469SB |